The objective of this proposal is to define the mechanisms regulating intracellular transport of lipids and proteins to myelin. One set of experiments characterizes transport of proteolipid protein (PLP) and sulfatide from their sites of synthesis to assembly into myelin or oligodendroglial (OL) plasma membrane. The hypothesis that PLP and sulfatide are co-transported will be investigated by comparing distribution and metabolism of PLP with sulfatide in intracellular pools. Sulfatide will be compared to PLP with regard to (1) sites of accumulation when transport is blocked by ionophores or agents which disrupt cytoskeleton: (2) orientation in intracellular vesicles and OL plasma membrane; and (3) long-term turnover in extra-myelin pools. These studies will utilize in vivo and in vitro labelling with (3H)leucine and (35S)sulfate, and subcellular fractionation of whole tissue and OL isolated from rat brainstem. Immunocytochemical staining of OL and their membrane sheets in culture will directly visualize these intracellular pools. The second set of experiments characterizes intracellular transport of galactocerebroside:Ab complexes in cultured OL and subsequent effects on OL membranes and metabolism. The effects of GalC Ab on OL will be compared to the effects of Ab to other glycolipids and the lectin peanut agglutinin. Ab to GalC in the presence of second Ab causes patching of GalC on OL membranes, and the GalC:Ab complexes are rapidly internalized. Sensitivity of these events to metabolic inhibitors, ionophores and agents which disrupt cytoskeleton will be examined. Metabolism of GalC will be measured by incorporation of (3H)galactose to determine if its synthesis is stimulated by internalization of GalC:Ab complexes. Exposure of OL to GalC Ab for 2-24 hours results in reversible condensation of membrane sheets; the role of cytoskeleton in this change will be investigated by immunocytochemical and biochemical methods. The hypothesis that continued exposure to GalC Ab alters proliferation and maturation of OL will be investigated by comparing incorporation of (3H)thymidine and time of appearance of GalC, 2'3'-CNP, basic protein and PLP in treated and control cultures. These studies are relevant to further understanding and eventual treatment of diseases involving myelin. Characterization of mechanisms controlling myelin assembly and OL function will provide significant information about regulation of myelination in normal development and remyelination following damage.